Determining the quantity and location of a molecule in a cell with a high degree of accuracy is a technically challenging process. The most common methods used to quantify cellular penetration of an exogenously added molecule include monitoring the uptake of dye-labeled molecules by microscopy or flow cytometry. Alternatively, cell penetration assays can include transcriptional readouts, quantitative mass spectrometry readouts, or dye-mediated methods that produce signals based on changing chemical environment.
These methods can be technically challenging, lack the spatial acuity to distinguish molecules that are trapped in endosomes from material in the cytosol, and are prone to additional artifacts, such as leakage of a fluorescent molecule during physical manipulation or after fixation and light-induced redistribution of signal from a fluorescent molecule. Furthermore, existing methods to quantify penetration of a molecule often require the molecule of interest to be conjugated to a bulky chemical group that perturbs the molecule's physical properties and molecular interactions, and/or require expensive instruments, such as microscopes for imaging. In nearly all cases, these methods cannot provide a highly quantitative measurement.
Accordingly, improved methods for identifying and quantifying molecules in cellular compartments are needed.